api.rb

# This is API module, mapped under /api

require 'sinatra/base'

module My
  class API < Sinatra::Base
    get '/' do
      'Happy API'
    end
  end
end

app.rb

# This is main app module, mapped under /, where I would like to define additional route in separate file 'fruit.rb' and loading it with load

require 'sinatra/base'

module My
  class App < Sinatra::Base
    get '/' do
      'Happy App'
    end

    load './fruit.rb'

  end
end

app.ru

# I run this with `shotgun -p 5000 app.ru` environment being `RACK_ENV=development`

require 'sinatra'

require './app' # the file with My::App class
require './api' # the file with My::API class

disable :run

map '/' do
  run My::App
end

map '/api' do
  run My::API
end

fruit.rb

# This gets loaded with load './fruit.rb'

get '/fruit' do
  'Happy Fruit'
end