Engineering the aveC gene to enhance the ratio of doramectin to its CHC‐B2 analogue produced in Streptomyces avermitilis

Abstract

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are major commercial products for parasite control in the fields of animal health, agriculture, and human infections. Historically, the avermectin analogue doramectin (CHC-B1), which is sold commercially as Dectomax is co-produced during fermentation with the undesired analogue CHC-B2 at a CHC-B2:CHC-B1 ratio of 1.6:1. Although the identification of the avermectin gene cluster has allowed for characterization of most of the biosynthetic pathway, the mechanism for determining the avermectin B2:B1 ratio remains unclear. The aveC gene, which has an essential role in avermectin biosynthesis, was inactivated by insertional inactivation and mutated by site-specific mutagenesis and error-prone PCR. Several unrelated mutations were identified that resulted in improved ratios of the desirable avermectin analogue CHC-B1, produced relative to the undesired CHC-B2 fermentation component. High-throughput (HTP) screening of cultures grown on solid-phase fermentation plates and analysis using electrospray mass spectrometry was implemented to significantly increase screening capability. An aveC gene with mutations that result in a 4-fold improvement in the ratio of doramectin to CHC-B2 was identified. Subsequent integration of the enhanced aveC gene into the chromosome of the S. avermitilis production strain demonstrates the successful engineering of a specific biosynthetic pathway gene to significantly improve fermentation productivity of a commercially important product.

New Approaches for Bioaugmentation as a Remediation Technology

“…80 While gene alteration has been applied more often for industrial or agricultural applications, 14,124,126,224 it can also be useful for bioremediation processes. 30,37,125,225 Traditionally, the gene of interest is first cloned into a vector for maintenance in a laboratory organism, such as E. coli, that is more amenable to laboratory growth and manipulation.…”

Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

“…Elimination of this unproductive enzymatic activity from S. tubercidicus R922 is an important goal in the further optimization of the biocatalyst, as other biocatalytic parameters of this strain make it the most desirable host for Ema CYP expression. The unforeseen and only partially understood influence of the surrogate screening hosts on the performance of this biocatalyst also emphasizes the need for using a relevant screening system as close to the final application as possible during molecular evolution programs (30,31) and reinforces the notion that in a complex whole-cell bioconversion, the entire system has to be analyzed and optimized.…”

Stereoselectivity of Isolated Dehydratase Domains of the Borrelidin Polyketide Synthase: Implications for cis Double Bond Formation

“…[9,26] Finally, we have previously shown, for both KR and ER domains, that the energetic balance between different stereochemical outcomes on a modular PKS is finely balanced, and can be readily perturbed by changes either in substrate structure [5,27,28] or in active site amino acids. [2,29] Work to synthesise the natural di-and triketide substrates for BorDH2 and BorDH3 respectively, as their ACP-bound thioesters, is therefore in progress.…”

“…For example, most double bonds in these compounds (whether retained or further reduced) have trans (E) geometry, arising from the syn dehydration of 3R alcohols (as in the analogous process in fatty acid synthesis) [5,6] but a significant fraction of polyketides have double bonds with the alternative cis (Z) configuration. In some cases, cis double bonds have been shown to arise from the action of exogenous enzymes, [7][8][9] but for other modular PKSs it has been proposed that cis double bonds arise by syn dehydration of a 3S alcohol by the DH domain. [10] This hypothesis implies that in such cases the double bond geometry is controlled by the stereochemical outcome of KR-catalysed ketoreduction, rather than directly by the DH itself, and so cisgenerating modules should contain KR domains predicted to dictate production of 3S alcohols.…”