Comparative genomics of alexander fleming’s original penicillium isolate (imi 15378) reveals sequence divergence of penicillin synthesis genes

ABSTRACT Antibiotics were derived originally from wild organisms and therefore understanding how these compounds evolve among different lineages might help with the design of new

antimicrobial drugs. We report the draft genome sequence of Alexander Fleming’s original fungal isolate behind the discovery of penicillin, now classified as _Penicillium rubens_ Biourge

(1923) (IMI 15378). We compare the structure of the genome and genes involved in penicillin synthesis with those in two ‘high producing’ industrial strains of _P. rubens_ and the closely

related species _P. nalgiovense_. The main effector genes for producing penicillin G (_pcbAB_, _pcbC_ and _penDE_) show amino acid divergence between the Fleming strain and both industrial

strains, whereas a suite of regulatory genes are conserved. Homologs of penicillin N effector genes _cefD1_ and _cefD2_ were also found and the latter displayed amino acid divergence between

the Fleming strain and industrial strains. The draft assemblies contain several partial duplications of penicillin-pathway genes in all three _P. rubens_ strains, to differing degrees,

which we hypothesise might be involved in regulation of the pathway. The two industrial strains are identical in sequence across all effector and regulatory genes but differ in duplication

of the _pcbAB_–_pcbC_–_penDE_ complex and partial duplication of fragments of regulatory genes. We conclude that evolution in the wild encompassed both sequence changes of the effector genes

and gene duplication, whereas human-mediated changes through mutagenesis and artificial selection led to duplication of the penicillin pathway genes. SIMILAR CONTENT BEING VIEWED BY OTHERS

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Clinical use of antibiotics has revolutionised treatment of bacterial infection. The characterization of penicillin1 followed from Alexander Fleming’s discovery of lysis and inhibition of

the growth of _Staphylococcus_ on petri dishes colonized by the fungus _Penicillium rubens_2 (named at the time as _P. notatum,_ and until recently as _P. chrysogenum_3,4). There followed a

golden age in the use of antibiotics to combat bacterial disease5. It was soon realized, however, that this might be short-lived, as more and more pathogenic bacteria evolved resistance to

these compounds6,7,8. The result is an arms race between clinical use of antibiotics and the evolution of resistance in target bacteria, which requires new classes of antibiotics and new

methods of delivery if medicine is to retain the upper hand. One potentially useful way to improve antibiotic use and deployment is to draw inspiration from the evolution of antibiotic

production and resistance in nature. Micro-organisms produce antibiotics to suppress the growth of their antagonists and reduce competition for space and resources9,10. Accordingly,

organisms are under selection to evolve resistance to the antibiotics of others, yet producers are under reciprocal selection to increase the efficacy of their own antibiotics against their

antagonists. With such an arms race11, it is expected that as antibiotic resistance evolves, antibiotics themselves will evolve too12. Genome sequencing can reveal variation in antibiotic

production pathways among organisms with different antibiotic profiles. For instance, the ability to produce new compounds might evolve by modification of the synthesis genes or metabolic

pathways, or there might be changes to existing compounds to counter the degradation of the antibiotic by resistant bacteria. These adaptations could encompass changes in the amino acid

sequence of effector genes, changes in regulators of the antibiotic-production pathway, or duplication of effector genes. Gene duplication might lead to increased expression of the enzyme if

the copies are conserved, or to production of multiple variants of the antibiotic if paralogous copies diverge in function13. In recent decades, considerable effort has gone into the study

of the evolution of antibiotic resistance as a solution to the crisis7,14, but the evolution of antibiotics themselves remains largely neglected. Understanding how antibiotics coevolve in

natural arms races with resistant bacteria might help to design methods for countering resistance evolution in the parallel arms race in clinical settings. In order to gain insights into the

evolution of genes underlying the production of the classic antibiotic, penicillin, we here present a draft genome sequence for Fleming’s original isolate, _Penicillium rubens_ (IMI 15378).

Cryopreserved living samples of this isolate are kept in numerous global collections, and we revived the fungus from the CABI (IMI) living culture collection for DNA extraction and

whole-genome sequencing. We compare the overall genome structure and variation in a set of genes involved in the penicillin-production pathway with closely related _Penicillium_ isolates

with sequenced genomes. In particular, we compare Fleming’s isolate to two industrial strains of _P. rubens_ derived from a second isolation from the wild in the USA. These strains were

originally misnamed as _P. chrysogenum_ (and still are in some public sequence databases) but were subsequently shown by multigene analysis to belong to the _P. rubens_ clade3,4. The

original wild US isolate (NRRL1951), isolated from a mouldy cantaloupe, was subjected to multiple rounds of X-ray, chemical (chlormethine) and ultraviolet mutagenesis and artificial

selection15 in order to generate isolates with high production rates of penicillin for industry16, including P2niaD18 and Wisconsin 54-125517. The two lines split after an initial shared

phase of both UV and X-ray mutagenesis and selection (via a common ancestor strain of Wis Q-176, see Fig. 1 in16,18,19). Wisconsin 54-1255 was derived from further rounds of UV and nitrogen

mustard mutagenesis and selection, while P2niaD18 was derived from a separate round of undocumented improvements by Panlabs Inc, followed by deletion of the nitrate reductase gene

niaD16,18,19. Any differences from the Fleming isolate that are shared by these two strains resulted either from evolution in the wild progenitors, or from the first steps of artificial

selection and mutagenesis in the lab. In contrast, differences between the two industrial strains are solely the result of mutagenesis and artificial selection for high production: for

example, comparison of P2niaD18 to the original Wisconsin 54-1255 genome17 revealed evidence for structural rearrangements and tandem duplication of penicillin-producing genes caused by the

mutagenesis20. To provide a broader context for evolution in the wild, we also compare the Fleming genome to another penicillin-producing species from the _Penicillium_ section _Chrysogena_,

_P. nalgiovense_21,22, Our focus is primarily on short-term evolution among the closely related strains rather than the longer-term acquisition of penicillin production and so we do not

repeat previous comparisons among more distantly related organisms23,24. As well as comparing genome structure, we searched for genes involved in the penicillin pathway and compared copy

number and sequence divergence among strains. Penicillin is a beta-lactam and encompasses several natural variations of the compound. _P. rubens_ produces penicillin G via a 15 kb gene

cluster of 3 genes (_pcbAB_, _pcbC_ and _penDE_) present in the genomes of various filamentous fungi25,26,27 (Fig. 1). The first two genes _pcbAB_ and _pcbC_ encode the enzymes

delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase (ACVS) and isopenicillin N synthase respectively23,28, which catalyse the formation of the first bioactive molecule in the

pathway, isopenicillin N26. All beta-lactams share this two-step pathway (including cephalosporins and cephamycins23,26,28). Evidence indicates that the genes _pcbAB_ and _pcbC_ were

horizontally acquired from bacteria by the beta-lactam producing fungi24. The third gene _penDE_ encodes the enzyme isopenicillin N acyltransferase, which catalyses the final step of the

biosynthetic pathway that synthesizes penicillin G27,29. This gene is hypothesized to have evolved within the beta-lactam producing fungi rather than being horizontally acquired from

bacteria24. In the beta-lactam producing fungi _Acremonium chrysogenum_, the genes _cefD1_ and _cefD2_ encode the isopenicillin N epimerase system and provide an alternative biosynthetic

pathway to produce penicillin N from isopenicillin N, and cephalosporin C from penicillin N30,31,32. Several genes have been identified that play a role in regulating the pathway leading to

penicillin G production, and evolved within beta-lactam producing fungi, described in more detail below24,33. We hypothesized that selection on antibiotic production should most likely

result in changes in the coding sequence or copy number of effector genes at later stages of the pathway (i.e._ penDE_, which is unique to the production of penicillin G as opposed to other

beta-lactams), or of the regulatory genes, rather than the upstream effector genes that generate pre-cursors used by multiple antibiotics. MATERIALS AND METHODS CULTURING, DNA EXTRACTION AND

SEQUENCING The fungus _Penicillium rubens_ Biourge (1923)34 IMI 15378 (= ATCC 8537; NRRL 824; CBS 205.57) was obtained from the CABI IMI culture collection. As part of a separate experiment

(not reported here), replicates of fungus were grown for 11 weeks at 20 °C on petri dishes with LB Lennox agar media with addition of 20 g/L of sucrose. Fungus from each of 6 treatments was

then cultured in LB Lennox Broth with 20 g/L of sucrose at room temperature for a week prior to the DNA extraction. For each of the six treatments, around 100 mg of washed mycelium was

ground under liquid nitrogen and DNA was extracted using the DNeasy Plant Mini Kit (Qiagen). DNA libraries were prepared with an Illumina TruSeq PCR-free kit at the Department of

Biochemistry, University of Cambridge, and sequenced with Illumina MiSeq v2 technology with 2 × 150 paired-end sequencing and 350 bp insert size. Separate library preparations and sequencing

were performed for the 6 separate extractions, but subsequent results indicated no changes had accrued among the treatments, so reads were pooled for the assembly and analyses presented

here. GENOME ASSEMBLY AND ANALYSIS Raw reads were filtered for low-quality bases and adapter sequences using BBTools ‘bbduk’ v38.22 (available at https://sourceforge.net/projects/bbmap/)

with the parameters ‘ktrim = r k = 23 mink = 11 hdist = 2 maq = 10 minlen = 100 tpe tbo’. An initial assembly was constructed using SPAdes v3.13.035 with default parameters and potential

contamination was assessed using BlobTools v1.036. Genome completeness was assessed using Benchmarking Universal Single-Copy Ortholog (BUSCO) gene sets v3.0.2 for Eukaryota (_n_ = 303) and

Fungi (_n_ = 290)37. Assembled genomes for the two industrial strains, P2niaD18 and Wisconsin 54-1255, and _P. nalgiovense_ (IBT 13039) were downloaded from NCBI GenBank (Table 1). The

P2niaD18 genome was scaffolded into whole chromosomes in the source paper by comparing alignments to the Wisconsin 54-1255 genome. Genomes were aligned using nucmer in the mummer package

4.0beta, and structural changes visualized with dotplots using the DNANexus Dot browser (available at https://dnanexus.github.io/dot/). All raw sequence data have been deposited in the

relevant International Nucleotide Sequence Database Collaboration (INSDC) databases under the Study ID PRJEB35151 (see Table S1 for run accessions). PENICILLIN PATHWAY GENES We searched for

the _pcbAB_, _pcbC_ and _penDE_ genes in each genome using BLAST, and for a paralog of _penDE_ that was first discovered in the Wisconsin 54-1255 genome17,38 and functionally

characterized39. Query sequences are listed in Table S2. We also searched for _cefD1_ and _cefD2_ genes, which catalyse an alternative pathway for converting isopenicillin N to penicillin N

rather than penicillin G, and were previously discovered in the Wisconsin 54-1255 genome17 and shown to be expressed. In addition, we searched for a suite of genes identified as playing a

regulatory role in penicillin production: _anBH1_, the three subunits of the transcription factor _ancF_ (_hapB_, _hapC_, and _hapE_), _pacC_, and _veA_. Functions of these genes are

summarised in Fig. 1. In brief, PacC is a wide domain pH and carbon source dependent regulator_,_ which upregulates the _pcbAB_ and _pcbC_ genes in _P. chrysogenum_ in an alkaline

environment and/or when the fungus is grown on a depleted carbon source40,41. VeA is a wide domain light dependent regulator in _P. chrysogenum_, _A. chrysogenum_ and _Aspergillus nidulans_.

It is involved in upregulation of _pcbAB_ and downregulation of _pcbC_26,42,43. The transcription factor ancF consists of 3 subunits hapB, hapC and hapE and is responsible for the

downregulation of the gene _pcbAB_ and upregulation of the genes _pcbC_ and _penDE_ in _P. chrysogenum_44,45,46. The _anBH1_ gene produces the basic-region helix loop helix protein (bHLH)

that binds to the promoter region upstream of _penDE_ and downregulates the transcription of _penDE_26,47. For each gene, we generated an alignment (including multiple copies where present)

using MAFFT 1.3.648 and reconstructed a phylogenetic tree by maximum likelihood using the GTR + invgamma model in PHYML 2.2.349, implemented in Geneious 9.1.8 (Biomatters Ltd, Auckland, New

Zealand, https://www.geneious.com). We tested for evidence of positive selection among strains by running codon models in PAML 450 for genes displaying variation: the null model of a single

dN/dS ratio across codons, referred to as ω; the neutral model with a fraction _p_1 of codons that are under purifying selection (dN/dS < 1; _ω_1) and a fraction _p_2 evolving neutrally

(dN/dS = 1; _ω_2); and the positive selection model including an additional fraction _p_3 codons evolving positively (dN/dS > 1; _ω_3). We used the Akaike Information Criterion to select

the best model while penalizing for differences in the number of free parameters (null = 1, neutral = 2, positive = 4). The test is conservative because it requires substantial changes in

amino acids to detect positive selection, whereas in reality a single amino acid change could underlie functional divergence. In addition to comparing best models, we plotted the average

dN/dS ratio across the genome (ω) as a measure of degree of amino acid conservation among strains, with low values indicating stronger overall purifying selection. RESULTS GENOME ASSEMBLY OF

_P. RUBENS_ (IMI 15378) A total of 2.86 Gb trimmed data (82.3% raw; Table S1) were used to generate an initial assembly comprising 274 scaffolds spanning 30.51 Mb. Screens for potential

contamination resulted in the removal of 6 scaffolds (15.7 kb) that were not marked as from the genus _Penicillium_ based on sequence similarity to NCBI ‘nt’ and UniRef90 public repositories

(Fig S1). Scaffolds less than 500 bp in length were also discarded, resulting in a final draft assembly of 101 scaffolds spanning 30.46 Mb total length (~ 94X coverage), with an N50

scaffold length of 1.62 Mb. Assembly quality, based on the presence of core eukaryotic and fungal genes (BUSCO), indicated a high level of gene completeness (99.0% and 99.3% respectively)

and a low level of duplication (0.3% and 1.0% respectively), suggesting a haploid genome assembly in common with the other strains (Table 2). The assembly is marginally smaller than the

assemblies of the two industrial strains (32.4 and 32.2 Mb respectively) and has similar GC content (48.9% vs 49% for both industrial strains). The final genome assembly for _P. rubens_ IMI

15378 (nPRUBv1) has been deposited at DDBJ/ENA/GenBank under the accession GCA_902636305.1 (CACPRF010000001–CACPRF010000101). STRUCTURAL COMPARISON AMONG PENICILLIUM GENOMES The genome of

Fleming’s _P. rubens_ (IMI 15378) is broadly colinear with the P2niaD18 genome that was assembled to whole chromosome level, with relatively few cases of translocation or transversions (Fig.

 2). More rearrangements are apparent between the Wisconsin 54-1255 strain and the P2niaD18 genome, perhaps indicative of structural mutations caused by mutagenesis during the improvement

process as previously reported20. The _P. nalgiovense_ IBT 13039 genome was broadly colinear with P2niaD18, although the greater fragmentation of the assembly makes it harder determine any

large-scale rearrangements. All three genomes of _P. rubens_ are highly similar at the sequence level: the Fleming genome is 0.106% divergent from the P2niaD18 genome across aligned regions

(31,547 SNPs from 29.7 Mb alignment) whereas the Wisconsin 54-1255 is only 0.0038% divergent (1231 SNPs from 32.2 Mb alignment). _P. nalgiovense_ IBT 13039 is 5.8% divergent (1,484,129 SNPs

from 24.8 Mb aligned regions). The structure of the penicillin effector genes is conserved across species and always falls into the well characterized cluster of _pcbAB_, _pcbC_ and _penDE_

genes (Fig. 3). The P2niaD18 genome alone has a single tandem duplication of the whole cluster, rather than multiple complete or partial tandem duplications of the cluster present in other

industrial penicillium strains20,51,52. In addition to the main loci, a partial duplicate exhibiting a match to the final 123 bp of _pcbAB_ but with three amino acid substitutions is found

in a non-coding region in the two industrial strains, 2704 bp downstream of _pcbAB_ (Fig. 3, Table S3). This fragment, labelled B1 in the previous analysis of Wisconsin 54-1255 by Fierro et

al_._52 is found in both tandem duplicates in P2niaD18, but absent from the Fleming genome (as confirmed by mapping raw reads of Fleming strain onto the Wisconsin 54-1255 genome, Fig. S2).

We speculate that this might play a functional role in the region, for example in regulating expression of _pcbAB_, but it might simply be a neutral or deleterious side-effect of the

mutagenesis during improvement of those strains. Other putative beta-lactam effector genes were found in the genomes of all the four strains compared. All four strains contained the paralog

of _penDE_ first identified in the Wisconsin 54-1255 genome17. The _cefD1_ region contains additional 36 to 57 bp long fragments that blast to genome regions outside the main coding region.

BLAST confirmed that these are not repeated domains or regions found elsewhere in the genome but represent single partial duplications similar to those observed in _pcbAB_. Two of these

duplicate fragments were found only in the three _P. rubens_ strains and two only in _P. nalgiovense_. The _cefD2_ region was not duplicated but recovered as two long sections and one short

section in all three genomes, indicating the absence of match across the full-length region found in the _P. arizonense_ gene used for the query. The genes involved in regulation of

penicillin production were scattered across the genome of each strain (Table S3). The _hapB_ gene in the _ancF_ transcription factor complex displays a partial duplicated fragment of 95 bp

in the two industrial strains, which is lacking in the other two genomes. A clear _hapE_ match was missing for _P. nalgiovense_. All other regulatory genes are present in single copy in all

four genomes. SEQUENCE DIVERGENCE OF PENICILLIN EFFECTOR AND REGULATORY GENES The two industrial strains, P2niaD18 and Wisconsin 54-1255, were identical at the sequence level for all the

focal genes and therefore for subsequent analyses only sequences from Wisconsin 54-1255 were used to represent the American isolate of _P. rubens_. In contrast, penicillin-pathway genes have

diverged in amino acid sequence between the Fleming strain and the US strains. All three effector genes encoding enzymes in the penicillin G pathway have diverged, but _pcbAB_ and _penDE_

showed the highest rates of amino acid divergence relative to silent changes whereas _pcbC_ was strongly conserved (Fig. 4, Table S4). The level of divergence in _pcbAB_ is unexpected since

this gene functions to produce the initial precursor in the pathway, which is shared in the production of other beta-lactams. The homologs of _cefD1_ and _cefD2_ genes were found in all

genomes of _P. rubens_ strains. This was unexpected as these genes are involved in the synthesis of the cephalosporin intermediate penicillin N in _A. chrysogenum_ and are not known to have

a functional role in _P. rubens_17,53,54. The penicillin N effector gene _cefD2_ also showed a high level of amino acid divergence whereas _cefD1_ was more strongly conserved than other

effector genes. The best sequence model for the effector genes plus _hapB_ was a model with most codons being under constraint (dN/dS < < 1) but with a significant proportion of codons

being unconstrained (dN/dS = 1). There was no sequence divergence between American and British _P. rubens_ isolates in the _penDE_ paralog, _pacC_, _ancF_ (_hapB_, _C_ and _E_) or _veA_. To

further investigate possible regulatory changes, we looked for sequence variation within transcription factor binding sites within the intergenic region between _pcbAB_ and _pcbC_, which is

a bidirectional promotor region for these genes. Among 28 binding sites previously identified in _P. chrysogenum_55, all were found in the Fleming genome, and just one site was lost in both

industrial strains (GATA to GGTA mutation, Table S5). Thus, the divergence of known binding sites is low, similar to that seen for regulatory proteins. DISCUSSION Nearly a century since

Alexander Fleming discovered the action of penicillin in bacterial cultures contaminated by _P. rubens_, we report the first draft genome sequence of his original strain. Very soon after the

original discovery and isolation of penicillin, a second wild isolate of _P. rubens_ from the USA was employed for future industrial manufacture owing to its greater rate of penicillin

production15. Consequently, two of the strains derived from this isolate have been the focus of previous whole genome sequencing within the _P. rubens_ clade16,17,20. We compared these

genomes with each other and a third, more distantly related genome of _P. nalgiovense_56. Comparison of the two USA strains provides insights into the industrial mutagenesis and artificial

selection process16, which was originally performed by selecting phenotypically useful mutants without knowledge of the underlying genomic basis15. There were no amino acid differences at

any genes encoding the enzymes in the penicillin pathway and regulatory genes. Instead, there was evidence for structural rearrangement across the genome, including tandem duplication of the

_pcbAB_–_pcbC_–_penDE_ cluster in P2niaD18, which has previously been studied in these and other industrial strains51,52. This fits with the type of mutagenesis and artificial selection

used for this process. Experimental work showed that tandem duplication of the _pcbAB_–_pcbC_–_penDE_ cluster does not directly increase penicillin production over short time periods—a

strain of P2niaD18 that was modified to lose one copy did not produce significantly less penicillin over a 96-h assay period57. Substantial copy number multiplication of the region among

industrial strains still seems to implicate gene duplication in penicillin production, but perhaps only under specific growth conditions or over longer periods18,51. Another plausible source

of variation would be changes in regulatory regions, but experimental evidence indicates that such variation is unlikely to contribute to increased penicillin production18. Comparison

between the UK and US genomes sheds light on both evolved differences between the wild progenitors of the strains, and potential initial changes in the domestication steps prior to the

divergence of P2niaD18 and Wisconsin 54-1255. One structural difference shared by the US genomes was the partial duplication of the final portion of the _pcbAB_ gene. Read mapping confirmed

that this region is missing from the Fleming genome and not just absent due to assembly artefacts (Fig. S2). Partial duplication and inversion have been documented previously at the ends of

the amplified region containing the penicillin synthesis genes for Wisconsin 54-125552. Furthermore, partial duplication has been found to play a role in generating novel diversity

previously, e.g., in the case of pathogen resistance in barley58, and could play a role in gene regulation. Without further sequencing, we cannot be certain whether this change occurred in

the wild progenitor of the US strains or during initial stages of domestication. Because of the nature of these changes in relation to the predicted effects of mutagenesis, however, and the

fact that further such differences arose between P2niaD18 and Wisconsin 54-1255, it seems plausible that shared structural differences of the two industrial strains from the Fleming genome

occurred during their initial shared history of mutagenesis prior to their separation. No sequence divergence was observed between the two US strains in any of the genes involved in

penicillin G production and regulation of the pathway: mutagenesis and selection for improved function resulted in major structural changes but no substitutions at these loci. In contrast,

penicillin-pathway enzymes have diverged in amino acid sequence between the Fleming strain and the US strains, especially _pcbAB_, _penDE_ and _cefD2_. While it is possible in principle that

these changes were caused by mutagenesis during domestication of the US strains, we think that this is unlikely: subsequent rounds of the same process led to no sequence divergence between

the US strains, and the numbers of substitutions involved would seem more commensurate with longer periods of time elapsing. Instead, these differences are likely to have accrued during

evolutionary divergence of the UK and US strains of _P. rubens_ in the wild. Although the level of divergence did not meet the statistical criteria for detecting significant evidence of

positive selection, a low level of constraint on protein sequence of these genes could still indicate a history of divergent selection at a subset of codons. Alternatively, it could indicate

that the function of these proteins is less dependent on amino acid identity at several sites than is the case for the other genes. In _A. nidulans_, the _aatA_ gene (an ortholog of

_penDE_) encodes the enzyme isopenicillin N acyltransferase26,59. It has been found that disruption of this gene does not disrupt penicillin production in _A. nidulans_. A paralog of _aatA_,

_aatB_ compensates for this as it encodes a homolog of isopenicillin N acyltransferase59. It should be noted that the isopenicillin N acyltransferase encoded by _aatA_ is only 55.2% similar

to its homolog encoded by _aatB_ and the two genes themselves are only 58% similar59. Additionally, the liquid chromatography–mass spectrometry (LC–MS) data for penicillin compounds

synthesized by either of the genes indicate unexplained significant peaks in proximity to the peaks representing standard penicillin V or penicillin G compounds synthesized by these genes.

These unexplained peaks could represent penicillin analogues synthesized by _aatB_ and _aatA_. It would be worthwhile to investigate further how the differences in penicillin effector genes

translate into altered function of the enzymes encoded, such as variation in the substrate specificity or efficiency of the enzymes60. Such variation in specificity of the enzymes could

result in synthesis of penicillin G analogues. Furthermore, presence of a _penDE_ paralog, and _cefD1_ and _cefD2_ homologs in all the genomes compared in this study suggest the possibility

that these genes encode homologs of isopenicillin N acyltransferase and isopenicillin N epimerase respectively17,39,53,61. These enzymes could potentially synthesize analogues of penicillin

G and penicillin N. Other beta-lactam gene variants such as homologs of the gene encoding 7-alpha-cephemmethoxylase subunit, _cmcJ_, have also been identified in the genome of _P.

chysogenum_17. Studies suggest that many of these gene variants are expressed but further work is needed to elucidate the functional importance of these genes, which is currently

unclear17,39,54. The biosynthesis of penicillin G in _P. chrysogenum_ and _P. rubens_ consists of a simple three gene pathway, but in certain bacteria such as _S. clavuligerus_, as many as

twelve genes can be involved in the synthesis of beta-lactams such as cephamycin C26,62. Much of what is known regarding the evolution of diversity of natural antibiotics stems from the

concept of rearrangement of genes in an existing biosynthetic gene cluster, or by addition of novel genes to existing clusters via processes such as horizontal gene transfer12,63. Our

analyses indicate that individual genes of beta-lactam biosynthetic pathways can themselves vary between species. Evidence indicates that many penicillin producing species such as _P.

chrysogenum_ are genetically diverse, and allelic variation within wild _P. chrysogenum_ populations can impact penicillin production within these populations64,65. Thus, it is plausible

that sequence variation in the genomes that we describe could account for the production of novel penicillin analogues. Subtle variation in chemical structure of antibiotics has been

identified for other antibiotics such as antimycins produced by _Streptomyces_63,66,67. Future work to sample variation more widely in _P. rubens_ and measure the impacts of variation on

chemical structure of penicillin compounds is needed to distinguish these alternatives. In conclusion, our results provide preliminary evidence that genes involved in the production of

penicillin display relatively high rates of amino acid divergence between populations, as predicted if antibiotics evolve in an arms race with antagonistic microbes. Moreover, the results

indicate that natural changes involving point mutation and amino acid substitutions were not fully explored by the classical industrial mutagenesis approach, which instead produced larger

structural rearrangements. Thus, the mutagenesis approach employed previously may have missed some solutions for optimizing penicillin design compared to natural selection in the wild,

especially in the context of robustness to evolving antibiotic resistance. Future approaches could use solutions explored by nature as a template for the development of novel antibiotic

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  Google Scholar  Download references ACKNOWLEDGEMENTS This work was funded in part by a Natural Environment Research Council Grant NE/S010866/1 and a project bursary from the Masters by

Research in Computational Methods in Ecology and Evolution at Imperial College London. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Life Sciences, Imperial College London,

Silwood Park Campus, Ascot, Berkshire, SL5 7PY, UK Ayush Pathak, Reuben W. Nowell, Christopher G. Wilson & Timothy G. Barraclough * Department of Zoology, University of Oxford, 11a

Mansfield Rd, Oxford, OX1 3SZ, UK Reuben W. Nowell, Christopher G. Wilson & Timothy G. Barraclough * CABI, Bakeham Lane, Egham, Surrey, TW20 9TY, UK Matthew J. Ryan Authors * Ayush

Pathak View author publications You can also search for this author inPubMed Google Scholar * Reuben W. Nowell View author publications You can also search for this author inPubMed Google

Scholar * Christopher G. Wilson View author publications You can also search for this author inPubMed Google Scholar * Matthew J. Ryan View author publications You can also search for this

author inPubMed Google Scholar * Timothy G. Barraclough View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS A.P. and T.G.B. conceived the

study. M.R. provided materials. A.P. and C.G.W. grew cultures and extracted DNA. A.P., R.W.N. and T.G.B. analysed the data. A.P. and T.G.B. led writing the manuscript with further inputs

from R.W.N., M.R. and C.G.W. We thank 3 anonymous reviewers for extremely helpful comments on earlier drafts. CORRESPONDING AUTHOR Correspondence to Timothy G. Barraclough. ETHICS

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Alexander Fleming’s original _Penicillium_ isolate (IMI 15378) reveals sequence divergence of penicillin synthesis genes. _Sci Rep_ 10, 15705 (2020).

https://doi.org/10.1038/s41598-020-72584-5 Download citation * Received: 13 November 2019 * Accepted: 03 September 2020 * Published: 24 September 2020 * DOI:

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