# Mechanisms and Evolution of Assembly-Line Polyketide Synthases

> **NIH NIH R35** · STANFORD UNIVERSITY · 2023 · $401,643

## Abstract

PROJECT DESCRIPTION
Assembly-line polyketide synthases (PKSs) are enzyme machines that catalyze vectorial biosynthesis of a
growing polyketide chain through a uniquely defined sequence of acyl carrier protein and ketosynthase
domains involving alternating chain translocation and elongation reactions. Notwithstanding the discovery of
>3000 naturally occurring assembly-line PKSs, we do not understand how they blend catalytic specificity with
evolutionary flexibility. Our lab is motivated by the goal of understanding the enzymology and evolution of
assembly-line PKSs while enhancing our ability to engineer known PKSs and decode “orphan” ones.
Our Goals for the next five years are to:
1) Understand the chemical logic of vectorial biosynthesis by an assembly-line PKS: We will study: (i) the
structural dynamics of individual PKS modules; (ii) how different conformations of a module enable its
elementary reactions; and (iii) the extent to which transitions between successive reactions are coordinated
across an assembly line. Our proposed mechanistic investigations will exploit: (i) our ability to functionally
reconstitute PKSs in vitro; (ii) epitope-specific monoclonal antibodies to trap individual PKS modules in
specific conformational or catalytic states; and (iii) advances in X-ray, SAXS, and cryoEM analysis of PKSs.
2) De-orphanize the nocardiosis-associated NOCAP synthase: We have de-orphanized the nonamodular
NOCAP synthase found in isolates of Nocardia associated with nocardiosis. Now, we propose to solve the
structure of the fully tailored natural product, and to elucidate its biological role in nocardiosis. This will require
us to: (i) characterize a putatively doubly glycosylated polyketide product; (ii) establish a phenotypic assay for
its bioactivity in a macrophage-like human cell line; and (ii) harness genome-wide CRISPR knockout and
shRNA knockdown screens to gain insight into its mode of action.
3) Decipher the role of GRINS in the evolution of assembly-line PKSs: We have discovered a new genetic
element, named GRINS (genetic repeats of intense nucleotide skews), that is widespread in assembly-line
PKS genes. We hypothesize that GRINS play a major role in diversifying assembly-line PKSs. To test this
hypothesis, we will: (i) identify candidate genes in Streptomyces that are involved in introducing nucleotide
skews or enabling gene conversion; (ii) identify a bacterial host in which gene conversion is enabled by GRINS
under laboratory conditions; and (iii) develop an experimental model for GRINS-based PKS engineering.
The significance of our proposal is two-fold. On one hand, it offers the opportunity to break new ground in our
understanding of the structure, mechanism, and evolution of assembly-line PKSs. On the other hand, it tests
the extent to which our understanding of these remarkable megasynthases can be harnessed to discover
novel bioactive polyketides from “orphan” assembly-line PKSs.

## Key facts

- **NIH application ID:** 10620652
- **Project number:** 5R35GM141799-03
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** CHAITAN KHOSLA
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $401,643
- **Award type:** 5
- **Project period:** 2021-06-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10620652

## Citation

> US National Institutes of Health, RePORTER application 10620652, Mechanisms and Evolution of Assembly-Line Polyketide Synthases (5R35GM141799-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10620652. Licensed CC0.

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